JP4467888B2 - Aloe-emodin derivatives and their use in the treatment of neoplastic pathology - Google Patents

Abstract

The invention relates to aloe-emodin (AE) derivatives and their use as anticancer drugs. Saids derivatives show a specific cytotoxicity to tumour cells, also of neuroectodermal origin, to which they may in particular act as aloe-emodin prodrugs. Said pharamcological profile makes them particularly suitable for use in the treatment of neoplasias. Thereofore, pharmaceutical compositions containing said compound may be usefully used in the treatment of neoplasias. It has surprisingly been found that aloe-emodin derivatives in position 3' (bearing either a positive or negaive charge) exhibit improved sollubility properties and, at the same time, in vitro show cytotoxicity to tumour cells, also of neuroectodermal origin.

Description

  The present invention relates to aloe-emodin (AE) derivatives and their use in the treatment of neoplastic pathology.

  As is well known, the therapeutic strategies employed to treat tumors are essentially aimed at killing all malignant cells at both the first site and the metastatic site. For this purpose, from surgery, radiation therapy for tumors confined to a well-defined area or organ, chemotherapy for local or systemic tumors, endocrine therapy for hormone-dependent tumors, Various therapies are used, up to immunotherapy and hyperthermia. All of the above methods are used alone or in combination depending on the tumor cell type and the stage of the disease in order to obtain an effective therapy for tumor cell eradication.

  The most common therapeutic approach is probably chemotherapy, used alone or in combination with the above therapies. The ideal chemotherapeutic agent should be selected for the tumor cells and should not produce serious adverse effects on normal cells as well as systematic toxic effects. However, despite the numerous research results that have been formed over several years to identify selective anticancer drugs, no compound has been used alone or in combination with other compounds, and a satisfactory therapeutic index, i.e. It has not been proven to possess a ratio between the effects on tumor cells and the absence of cytotoxic effects on non-malignant cells. There are many anticancer agents that are known and in clinical use, and many are mechanisms based on cytotoxicity against tumor cells. Alkylating drugs, such as nitrogen mustard, should be used first, followed by antimetabolite drugs, folate-antagonists such as methotrexate, or purine-antagonists such as 6-mercaptopurine, 5 -Pyrimidine-antagonists such as fluorouracil, vegetable-derived cell mitotic blockers such as vincristine, vinblastine, and podophyllotoxin, antibiotics such as mitomycin, anthracycline and bleomycin, nitrosourea, Platinum coordination compounds, and recently so-called biological response modifiers such as α-interferon and enzymes such as asparaginase are used. All of the above drugs are used extensively, alone or in combination, in a wide range of tumors ranging from tumors located in limited organs to systematic tumors. For example, for neuroectodermal-derived tumors such as neuroblastoma, peripheral primitive neuroectodermal tumor (PNET), Ewing sarcoma, melanoma, microcytoma, etc., the commonly used chemotherapeutic agents are For example, but not limited to, vincristine and vinblastine, platinum coordination compounds and other compounds suitable for this purpose.

  Despite the widely recognized effects of many of the compounds described above, none have proved to have the ideal profile described above. In fact, multiple resistance of tumor cells to the drug is often seen, while toxic effects on other cells are being created.

In view of identifying compounds that can selectively act on tumor cells without inducing serious or general toxic effects on other proliferating cells, Applicants have identified the natural compound, aloe-emodin. again attention to, but itself has certain cytotoxic to tumor cells derived from neuroectodermal found not to induce severe toxic effects (the Italian patent application No.MI2000A001216 (WO 01/91735 pamphlet)) ).

  Aloe-emodin (AE) suffers from the disadvantage that it hardly dissolves in water or physiological solution, while only in water alkalized with heated alcohol, ether, benzene and ammonia or acidified with sulfuric acid. Can dissolve.

  Therefore, from the standpoint of pharmaceutical practice, the above-mentioned characteristics are damaged, making it difficult to prepare a pharmaceutical product effective for treatment.

Abrahamson, HN, et al. (J. Med. Chem. 1986, 29, 1709-1714) describe derivatives of aloe-emodin and glucosamine, without disclosing the antitumor activity of water-soluble model compounds. Have gained.

  Therefore, it is an object of the present invention to provide an aloe-emodin (AE) derivative that exhibits improved solubility while maintaining bioactivity similar to AE and potentially being an AE prodrug. There is to do.

  Surprisingly, the 3-position aloe-emodin derivative (positively or negatively charged) exhibits improved solubility, while at the same time in vitro and against neuronal ectoderm-derived tumor cells. It was found to be toxic.

  Therefore, the object of the present invention is to formula (1)

An aloe-emodin derivative of
In formula (1), R is a saturated or unsaturated C ₂ -C ₆ linear or branched, linear aliphatic polycarboxylic acid, or an aryl polycarboxylic acid or an acetal of an amino acid or amino sugar or It may be a free radical composed of an inorganic acid group.

  Another object of the invention is the use of said aloe-emodin derivative for the treatment of neoplastic pathology and in a pharmaceutical composition derived from neuroectodermal.

  It is a further object of the present invention to provide a pharmaceutical composition containing as an active ingredient the aforementioned derivative suitable for the treatment of neoplastic pathology.

  The features and advantages of the present invention may be better understood from the following detailed description.

In the aloe-emodin derivative of formula (1), the free radical R on the 3-position hydroxyl group is:
A saturated or unsaturated aliphatic polycarboxylic acid having a linear or branched chain of 2 to 6 carbon atoms, substituted on the aliphatic chain with a suitable hydrophilic group; When R is a fatty acid of the group a), the acid is preferably (i) linear, such as oxalic acid, malic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, diglycolic acid. (Ii) or a branched acid; (iii) or an unsaturated acid such as maleic acid; (iv) or a tricarboxylic acid such as citric acid Be;
For example, an aryl polycarboxylic acid substituted on the aromatic ring with a small hydrophilic residue, such as OH, CH ₂ OH and the like; when R is an aromatic acid of group b), the acid is Preferably composed of an aromatic system containing at least one ring, if any, a hydrophilic substituent, and at least two carboxyl groups, such as phthalic acid and 1,2,4-benzenetricarboxylic acid Be selected from a group;
Amino acids (having an amino group at the α-position or other site) such as, for example, alanine, isoleucine, tyrosine, tryptophan and GABA;
Acetals with amino sugars, such as daunosamine and equivalents , excluding glucosamine ;
For example, residues of inorganic acids such as phosphoric acid and equivalents.

  Furthermore, in the derivatives forming the object of the present invention, when R belongs to the groups a) and b), the non-esterified carboxyl group takes the free form or the salified form; Possible and known counter ions are used, in particular sodium or potassium.

  In the derivatives forming the object of the present invention, when R belongs to the groups c) and d), the amino group takes the free or salified form; in the latter case, a pharmaceutically acceptable and known anionic group Are used, with acetate, trifluoroacetate, nitrate, chloride, bromide and sulfate being particularly preferred.

  The biological characteristics of the compounds synthesized according to the following examples and the present invention are transmitted without limitation by the therapy of the present invention.

(Ester of AE and maleic anhydride)
Mono- (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylmethyl) ester AE (51.7 mg; 270.2 g / mol) other than 2-ene-dioic acid 0.19 mmol), maleic anhydride (60.0 mg; 98.1 g / mol; 0.61 mmol) and DMAP (2.7 mg; 122.2 g; 0.022 mmol) in anhydrous tetrahydrofuran ( 5 ml). The reaction was carried out under a nitrogen stream and stirred at reflux temperature for 48 hours. The solvent was then evaporated at room temperature under vacuum.

  The reaction residue was purified by chromatography (eluent: 55% petroleum ether, 45% ethyl acetate).

7 mg of pure compound was obtained. ¹ H-NMR (DMSO _d6 ): 5.35 ppm (2 H, s, CH ₂ ); 6.45 ppm (1 H, d, (J = 14.8), ═CH); 6.55 ppm ( 1 H, d (J = 14.8) = CH); 7.43 ppm (2 H, m); 7.81 ppm (3 H, m, H ⁴ , H ⁵ , H ⁶ ); 12.05 ppm (2H, 2s, 2OH). 368 (M ⁺ ).

(Ester of AE and succinic anhydride)
Succinic acid mono- (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylmethyl) ester AE (51.7 mg; 0.19 mmol), succinic anhydride (61.0 mg 0.61 mmol) and DMAP (2.7 mg; 0.022 mmol) were dissolved in anhydrous THF (5 ml). The reaction was carried out under a nitrogen stream and stirred at reflux temperature for 48 hours. The solvent was evaporated at room temperature under vacuum. The residue was washed 3 times with ethyl ether. 8.8 mg of pure compound was obtained. ¹ H-NMR (DMSO _d6 ): 5.35 ppm (2 H, s, CH ₂ ); 2.55 ppm (2 H, m, CH ₂ ¹ ); 2.68 ppm (2 H, m, CH ₂₎ ¹ ); 7.35 ppm (2 H, m, H ² , H ⁷ ); 7.77 ppm (3 H, m, H ⁴ , H ⁵ , H ⁶ ). 370 (M ⁺ ).

(Ester of AE and diglycolic anhydride)
Acetic acid (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylmethoxycarbonylmethoxy) ester AE (50.0 mg; 0.185 mmol), diglycolic anhydride (25.8) mg; 0.22 mmol) and DMAP (2.3 mg; 0.18 mmol) were dissolved in anhydrous THF (5 ml). The reaction was carried out under a nitrogen stream and stirred at reflux temperature for 12 hours. The solvent was evaporated at room temperature under vacuum.

  The solid residue was dissolved in ethyl acetate. The organic solution was extracted with aqueous potassium bicarbonate. The aqueous solution was slightly acidified with acetic acid and extracted again with ethyl acetate. The organic solution was dehydrated with sodium sulfate. The solvent was evaporated under vacuum. 32 mg of pure ester was obtained.

¹ H-NMR (DMSO _d6 ): 5.3 ppm (2 H, s, CH ₂ ); 4.3 ppm (2 H, s, CH ₂ ¹ ); 4.16 ppm (2 H, s, CH ₂₎ ² ); 7.43 ppm (2 H, m, H ² , H ⁷ ); 7.81 ppm (3 H, m, H ⁴ , H ⁵ , H ⁶ ); 11.99 ppm (2 H, s ( Wide) OH ¹ , OH ⁸ ). 386 (M ⁺ ).

(Ester of AE and phthalic anhydride)
Phthalic acid mono- (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylmethyl) ester AE (50.0 mg; 0.185 mmol), phthalic anhydride (32.9 mg) 0.22 mmol) and DMAP (2.3 mg; 0.02 mmol) were dissolved in anhydrous THF (5 ml). The reaction was carried out under a nitrogen stream and stirred at reflux temperature for 12 hours. The solvent was evaporated at room temperature under vacuum. The reaction residue was purified by chromatography (eluent: 40% petroleum ether, 60% ethyl acetate). 9.3 mg of pure compound was obtained.

¹ H-NMR (DMSO _d6 ): 5.4 ppm (2 H, s, CH ₂ ); 7.1-8.0 ppm (9 H, m, H ² , H ⁷ ; H ⁴ , H ⁵ , H ^{6; H 2, H 3,} H 4, H 5). 418 (M ^<+> ).

(Ester of AE with 1,2,4-benzenetricarboxylic anhydride)
1,2,4-benzenetricarboxylic acid 1- (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylmethyl) ester AE (50.0 mg; 0.185 mmol), anhydrous Benzenetricarboxylic acid (42.7 mg; 0.22 mmol) and DMAP (2.3 mg; 0.02 mmol) were dissolved in anhydrous THF (5 ml). The reaction was carried out under a nitrogen stream and stirred at reflux temperature for 12 hours. The solvent was evaporated at room temperature under vacuum.

  The solid residue was dissolved in ethyl acetate. The organic solution was extracted with aqueous potassium bicarbonate. The aqueous solution was slightly acidified with acetic acid and extracted again with ethyl acetate. The organic solution was dehydrated with sodium sulfate. The solvent was evaporated under vacuum. 23 mg of pure ester was obtained.

¹ H-NMR (DMSO _d6 ): 5.47 ppm (2 H, s, CH ₂ ); 7.39-8.4 ppm (8 H, m, H ² , H ⁷ ; H ⁴ , H ⁵ , H ⁶ ; H ³ , H ⁵ , H ⁶ ); 11.95 ppm (2 H, s (wide) OH ¹ , OH ⁸ ). 462 (M ⁺ ).

(Ester of AE and tryptophan)
AE (50.0 mg; 0.185 mmol), Fmoc-L-tryptophan-OPfp (131.5 mg; 0.22 mmol) and DMAP (22.6 mg; 0.02 mmol) were dissolved in anhydrous THF. The mixture was refluxed overnight under a nitrogen stream. The solvent was evaporated under vacuum at room temperature. An ester of the protected amino acid was obtained.

The residue was collected with CH ₂ Cl ₂ (5 ml) and treated with piperidine (157.5 mg; 0.19 mmol) at room temperature for 1 hour.

The solvent and piperidine were removed under vacuum. The residue was collected with CH ₂ Cl ₂ (1 ml) and precipitated with hexane. The solid was washed twice with hexane (2 ml) and dissolved in ethyl acetate. The organic solution was extracted with aqueous potassium bicarbonate. The aqueous solution was slightly acidified and extracted again with ethyl acetate. The organic solution was dehydrated with magnesium sulfate. The solvent was evaporated under vacuum to give 8.5 mg of pure product.

¹ H-NMR (DMSO _d6 ): 5.15 ppm (2 H, s, CH ₂ ); 3.27 ppm (1 H, m, CH ¹ ); 4.0 ppm (2 H, t, CH ^{2 2} ) 7.05-7.91 ppm (9 H, m, H ² , H ⁷ ; H ⁴ , H ⁵ , H ⁶ ); 456 (M ⁺ ).

(Ester of AE and alanine)
To a solution of AE (100 mg; 0.37 mmol) in N, N-dimethylformamide (10 ml), triethylamine (0.1 ml; 0.74 mmol), DMAP (4.5 mg; 0.037 mol) and Boc-L-Ala-OSu (212 mg; 0.74 mmol) was added. After 16 hours at 60 ° C., water was added to obtain a precipitate. The mixture was filtered. The precipitate was first washed with water and then with cold diethyl ether. 67 mg (41%) of Boc-L-Ala-Aloe-emodin was obtained.

¹ H-NMR (DMSO _d6 ): 11.93 ppm (1 H, s, OH phenol); 11.91 ppm (1 H, s, OH phenol); 7.82 ppm (1 H, t, H ⁶ ) 7.70 ppm (1 H, d, H ⁵ ); 7.69 ppm (1 H, s, H ⁴ ); 7.39 ppm (1 H, d, H ⁷ ); 7.35 ppm (1 H , S, H ² ); 5.28 ppm (1 H, d, CH—O); 5.24 ppm (1 H, d, CH—O); 4.13 ppm (1 H, m, CH—N) ); 1.37 ppm (9 H, s, C (CH ₃ ) ₃ ); 1.29 ppm (3 H, d, CH ₃ ).

  Boc-L-Ala-AE (20 mg) was dissolved in a 20 ml centrifuge tube containing a 90% aqueous solution of TFA (2.0 ml) and stirred for about 1 hour.

  Cold ether (5 ml) was added to precipitate the product, separated from the supernatant by centrifugation and washed twice with cold ether.

  The obtained powder was dried to obtain 18 mg (87%) of L-Ala-AE.

¹ H-NMR: in D ₂ O: 7.53 ppm (1, t, H ⁷ ); 7.39 ppm (1 H, d, H ⁵ ); 7.33 ppm (1 H, s, H ⁴ ) 7.11 ppm (1 H, d, H ⁷ ); 7.03 ppm (1 H, s, H ² ); 5.16 ppm (2 H, s, CH ₂ —O); 4.19 ppm 1 H, m, CH—N); 1.53 ppm (3 H, d, CH ₃ ). 342 (M ^<+> ).

(Ester of AE and isoleucine)
Fmoc-L-Ile-OPfp (211 mg; 0.407 mmol) was added to a THF solution (5 ml) of AE (50 mg; 0.185 mmol). The solution was refluxed for 48 hours and the solvent was evaporated. The solvent was purified by chromatography (petroleum ether / ethyl acetate 3: 1). 42 mg (37%) of Fmoc-L-Ile-Aloe-emodin was isolated as an orange powder.

¹ H-NMR (DMSO _d6 ): 11.88 ppm (1 H, s, OH phenol); 11.86 ppm (1 H, s, OH phenol); 7.89-7.60 ppm (7 H, m , H ⁶ , H ⁵ , H ⁴ , H _α Fmoc aromatic proton); 7.39 ppm (1 H, d, H ⁷ ); 7.35 ppm (1 H, s, H ² ); 7.34- 7.25 ppm (4 H, m, H β Fmoc aromatic protons); 5.32 ppm (1 H, d, CH-O); 5.22 ppm (1 H, d, CH-O); 4. 35 ppm (1 H, m, CH—N); 4.16-4.05 ppm (CH _{2 in} 3 H, m, CH, Fmoc); 1.89 ppm (1 H, m, CH—CH ₃ ) _{; 1.43 ppm (1 H, m} , CH 2 -CH 3 in H _{; 1.26 ppm (1 H, m} , H of _{CH 2 -CH 3); 0.90 ppm} (3 H, d, CH-CH 3); 0.85 ppm (3 H, t, CH 2 -CH ₃ ).

  Fmoc-L-Ile-AE (19 mg; 0.0314 mmol) was treated with piperidine (0.05 ml; 0.502 mmol) in dichloromethane (2 ml) over 1 hour. The solvent and piperidine were removed under vacuum. The residue was suspended in n-hexane and centrifuged. After removing the supernatant solvent, the resulting solid was treated with HCl in ethanol (2 ml). Solvent evaporation gave L-Ile-AE × HCl (10 mg; 77%).

¹ H-NMR (methanol _d4 ): 11.88 ppm (2 H, s, OH phenol); 7.83 ppm (1 H, t, H ⁶ ); 7.75 ppm (1 H, s, H ⁴ ) 7.73 ppm (1 H, d, H ⁵ ); 7.45 ppm (1 H, s, H ² ); 7.41 ppm (1 H, d, H ⁷ ); 5.40 ppm (2 H , S, CH ₂ —O); 4.20 ppm (1 H, d, CH—N); 2.19 ppm (1 H, m, CH—CH ₃ ); 1.59 ppm (1 H, m, CH ₂ CH of H); 1.42 ppm (1, m, H of _{CH 2 CH 3); 1.09 ppm} (3 H, d, CH-CH 3); 1.05 ppm (3 H, t, CH ₂ -CH _3). 383 (M ⁺ ).

(Ester of AE and tyrosine)
To a solution of AE (100 mg; 0.37 mmol) in N, N-dimethylformamide (10 ml), triethylamine (0.1 ml; 0.74 mmol), DMAP (4.5 mg; 0.037 mol) and Boc-L-Tyr-OSu (280 mg; 0.74 mmol) was added. After 14 hours at 60 ° C., water was added to obtain a precipitate. The mixture was filtered. The precipitate was first washed with water and then with cold diethyl ether. 77 mg (19%) of Boc-L-Tyr-aloe-emodin was obtained.

_{^{1 H-NMR (DMSO d6)}} : 11.81 ppm (1 H, s, OH phenol); 11.76 ppm (1 H, s, OH phenol); 9.01 ppm (1 H, s, OH phenolic 7.83 ppm (1 H, t, H ⁶ ); 7.74 ppm (1 H, d, H ⁵ ); 7.64 ppm (1 H, s, H ⁴ ); 7.41 ppm (Tyr); 1 H, d, H ⁷ ); 7.35 ppm (1 H, s, H ² ); 7.01 ppm (2 H, d, J = 8.1 Hz, Harom Tyr); 6.01 ppm (2 H, d, J = 8.1 Hz, Harom Tyr); 5.28 ppm (1 H, d, CH—O); 5.24 ppm (1 H, d, CH—O); 4.13 ppm ( 1 H, m, CH-N); 1.37 ppm ( _{H, s, C (CH 3} ) 3).

  Boc-L-Tyr-AE (20 mg) was dissolved in a 20 ml centrifuge tube containing a 90% aqueous solution of TFA (2.0 ml) and stirred for about 1 hour.

  Cold ether (5 ml) was added to precipitate the product, the supernatant was separated by centrifugation and washed twice with cold ether.

  The obtained powder was dried to obtain 18 mg (87%) of L-Tyr-AE.

¹ H-NMR: in D ₂ O: 7.50 ppm (1 H, t, H ⁷ ); 7.41 ppm (1 H, d, H ⁵ ); 7.32 ppm (1 H, s, H ^{4 ); 7.10 ppm (1 H,} d, H 7); 7.02 ppm (1 H, s, H 2); 7.05 ppm (2 H, d, J = 8.1 Hz, Harom Tyr) 6.01 ppm (2 H, d, J = 8.1 Hz, Harom Tyr); 5.16 ppm (2 H, s, CH ₂ —O); 4.19 ppm (1 H, m, CH—); N). 433 (M ⁺ ).

(Ester of AE and alanine, salified with trifluoroacetic acid)
The compound of Example 7 (5 mg) was treated with concentrated trifluoroacetic acid (200 μl) and diluted with acetone. ala-Aloe-emodin trifluoroacetate was isolated, filtered and dried.

(Ester of AE and alanine, salified with hydrochloric acid)
The compound of Example 7 (5 mg) was treated with concentrated hydrochloric acid (300 μl) and diluted with acetone. Ala-Aloe-emodin hydrochloride was isolated, filtered and dried.

(Ester of AE and phosphoric acid)
Dibenzyloxyphosphoryl chloride (396.69 g / mol, prepared by a known method, 161 mg; 0.407 mmol) was added to a solution of AE (50 mg; 0.185 mmol) in THF (5 ml). did. After the solution was refluxed for 6 hours, the solvent was evaporated. The residue was purified by chromatography (petroleum ether / ethyl acetate 3: 1). The benzyl group was removed by catalytic catalytic hydrogenation in dilute sodium hydroxide at atmospheric pressure over Pd / C. 42 mg (64%) of PO ₃ ² --Aloe-emodin was obtained as an orange powder.

(AE acetal with daunosamine)
A solution of N, O-di (trifluoroacetyl) daunosaminyl-2-bromide (402.09 g / mol, prepared in a known manner, 160 mg; 0.4 mmol) in dichloromethane (3 ml) was oxidized. Reaction with AE solution (50 mg; 0.185 mmol) in anhydrous THF (3 ml) in the presence of mercury, mercuric dibromide and molecular sieves. When the reaction was in equilibrium (on chromatographic basis), the salt and molecular sieve were filtered and the solvent was evaporated under reduced pressure. The OH group and NH ₂ protecting group (trifluoroacetate) of the sugar were removed by methanolysis. The product was purified by chromatography on neutral alumina. 8 mg (0.02 mmol; 10%) of the chromatographically pure product was obtained.

¹ H-NMR (DMSO _d6 ): 1.15 ppm (3 H, d, 6.5 Hz, CH ₃ Dauno); 1.77 ppm (1 H, m, H ₂ Dauno); 3.31 ppm (1 H, m, H ₃ dauno); 3.62 ppm (1 H, m, H ₄ dauno); 4.14 ppm (1 H, m, H ₅ dauno); 5.25 ppm (1 H, broad singlet) , H ₁ Dauno); 5.35 ppm (2 H, s, CH ₂ ); 6.45 ppm (1 H, d, (J = 14.8), ═CH); 6.55 ppm (1 H, d, (J = 14.8), = CH); 7.43 ppm (2 H, m); 7.81 ppm (3 H, m, H ⁴ , H ⁵ , H ⁶ ); 11.99 ppm 12.05 ppm (2 H, 2 s, 2 OH). 399 (M ⁺ ).

In vitro biological quantification In vitro cytotoxicity quantification was performed on various tumor cell lines derived from neuroectodermal. The purpose of this quantification was also to investigate compound cytotoxic effects and their particularities. The cell lines used were human, especially neuroectodermal tumors: neuroblastoma (IMR-5, SJ-N-KP)
Tumors derived from others: colon adenocarcinoma (Lovo 109) and glioblastoma (A172).

The above cells were quantified with the following compounds:
AM: Maleic acid ester (ex. 1) dissolved in DMSO
AS: Succinate dissolved in DMSO (ex.2)
ADG: Diglycolate dissolved in DMSO (ex. 3)
AF: Phthalic acid ester (ex. 4) dissolved in DMSO
ABT: ester of AE and 1,2,4-benzenetricarboxylic acid dissolved in HEPES (N-2-hydroxyethylpiperazine-N-2 sulfonesulfuric acid) TRIP: tryptophan ester dissolved in DMSO (ex. 6)
ALA-NH2: ester of non-alanine alanine and a free amino group dissolved in DMSO (ex. 7)
PJ8: ester of isoleucine hydrochloride dissolved in DMSO (ex.8)
TIR-NH2: tyrosine non-chlorinated ester (ex. 9) dissolved in DMSO
EM9: alanine ester salified with trifluoroacetic acid dissolved in DMSO (ex. 10)
ALA: alanine ester (ex. 11) in the form of hydrochloric acid dissolved in an aqueous solution (HEPES 1M)
ALA-HCl: alanine ester in the form of hydrochloric acid dissolved in DMSO (ex. 11)
AEP: AE phosphate dissolved in HEPES (ex. 12)
AED: acetal (ex. 13) with AE and daunosamine dissolved in HEPES.

  Cells cultured by known methods were seeded 24 hours prior to treatment with various derivatives at a concentration of 1000 cells / 100 μl. As a molecule dissolved in dimethyl sulfoxide and DMSO, the concentration of the initial strain was 200 mM, and as a molecule dissolved in the HEPES buffer and physiological solution, the concentration was 1 mM. The scalar concentrations of the various aloe-emodin derivatives were 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM that cells in exponential growth phase were exposed for 72 hours. .

  For each quantification, control was performed with medium alone (RPMI with 10% inactivated fetal calf serum, FBS and 1% penicillin / streptomycin).

  The effect of each compound was expressed as an EC50 compared to the untreated control, ie, the effective concentration that allowed the presence of 50% viable cells. Cell viability was assessed by MTT dye quantification.

Results The following table shows the results obtained. EC50 values (μM) are provided for each compound and cell line. The compound marked with an asterisk represents the data obtained by the cells in a medium with inactivated FBS in order to obtain a large amount of esterase that splits the ester bond on the AE molecule.

  What is clearly shown from the above results is that the compound in question, which is derived from neuroectodermal cells, is a cytotoxic cell or tumor cell. In particular, the compound according to the present invention is adenocarcinoma Lovo 109. It is extremely active against tumor cells.

  Conversely, the compounds of the invention are less active against the glioblastoma A172 cell line (data not reported).

  Furthermore, the above experimental data shows that the derivative in question is an aloe-emodin prodrug. In tests under experimental conditions assuming non-inactivated FBS, and therefore in the presence of esterases that cleave ester bonds, the compounds in question show up to 6-fold potentiation.

  Subsequently, these compounds are advantageously used as chemotherapeutic agents in the treatment of tumors derived from neuroectodermal. The reason is to use aloe-emodin within a short period and in the presence of inactivated and non-inactivated serum (the latter conditions mimic physiological conditions). In fact, it has been found that aloe-emodin exerts a remarkable, in vitro and in vivo pharmaceutical effect specific to the cells without causing a general toxic effect.

  Thus, aloe-emodin derivatives, like those derived from neuroectodermal (eg, neuroblastoma, peripheral primitive neuroectodermal tumor (pPNET), Ewing sarcoma, melanoma, microcytoma, etc.) It is a preferred compound suitable for the treatment of general-purpose tumors. Tumors derived from neuroectodermal, ie, neuroblastoma, is one of the most common solid tumors in children, reaching 10% of childhood cancers, with an unfavorable prognosis in 85% of cases, and 100 This is extremely important considering that every ten new cases are diagnosed annually in Australia and 10-15 new cases are diagnosed annually in Europe and the United States.

  For this purpose, the derivatives according to the invention can be used for the preparation of pharmaceutical compositions, as well as formulations for topical administration, possibly at the first and / or second site of the tumor by the parenteral route and the oral route. Depending on the route, it can be administered in the form of pharmaceutical preparations commonly used for drug administration. Furthermore, the aloe-emodin derivative can be used for the preparation of a pharmaceutical composition suitable for the clearing action in autologous bone marrow transplantation.

  Any pharmaceutically acceptable excipient can be used to prepare the compositions of the present invention, including controlled release topical dosage vehicles or devices.

  A composition for treating tumors is in particular a formulation comprising an aloe-emodin derivative as an active ingredient and having an active ingredient content suitable for obtaining a therapeutic effect according to the object of the present invention. In particular, the dosage is, for example, in the range of 5 to 500 mg for unit dosage and 2 to 500 mg for cleansing action.

  The above-mentioned preparations can be prepared by a method known in the art or by a novel pharmaceutical technique using a pharmaceutically acceptable excipient, diluent, emulsifier, aqueous or oily polymer vehicle and the like. The formulation can release the active ingredient at a controlled rate, for example, a fast or sustained or delayed release rate.

  Formulations that can be dispensed by parenteral methods contain lyophilized products that contain suitable suspensions in aqueous or oily vehicles, or in buffered solutions, or are reconstituted immediately prior to dosing. It can be manufactured in all forms of classic pharmaceutical forms, such as ampoules.

  Formulations for oral administration are tablets, soft or hard gelatin capsules, oily or covered, pills, powders, suspensions and emulsions for reconstitution.

  The composition of the present invention is also suitable for applying an active ingredient to a first tumor site or a second tumor site and consists of a formulation suitable for topical or transdermal use in a vehicle or device. Is done.

  The excipients, binders, lubricants, anti-caking agents, etc. may be of any type and in any manner suitable for pharmaceutical purposes and suitable for the purposes of the present invention. For example, these are only polymeric excipients known and used in the pharmaceutical field, i.e. silica, talc, magnesium oxide, stearate, polyethylene glycol, gum arabic, polyvinylpyrrolidone and polyvinyl alcohol. Alternatively, it may be a natural polysaccharide such as sugar (eg mannitol, lactose, glucose, sucrose, fructose), cellulose and its derivatives, eg methylcellulose, carboxymethylcellulose, starch and alginate.

Claims (11)

Formula (1)
A compound represented by
In the formula (1), the R is
- saturated or unsaturated C ₂ -C _6, the linear or branched, and oxalic acid, malic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, diglycolic acid and citric acid A chain aliphatic polycarboxylic acid substituted on an aliphatic chain selected from the group consisting of :
An aryl polycarboxylic acid substituted on an aromatic ring selected from the group consisting of phthalic acid and 1,2,4-benzenetricarboxylic acid;
-An amino acid selected from the group consisting of alanine, isoleucine, tyrosine, tryptophan and 4-aminobutanoic acid, which is an amino acid having an amino group at the α-position or other site ;
Said compound which is a free radical of either an acetal of the amino sugar daunosamine ; or an inorganic acid composed of phosphoric acid. The free compound of claim 1 which release carboxyl groups are salified with possible counterions acceptance as a medicament. The compound according to claim 2 , wherein the counter ion is sodium or potassium. A compound according to claim 1, the free release amino groups are salified with possible anionic groups accepted pharmaceutically. Anion groups, acetate, trifluoroacetate, nitrate, chloride salt compound of claim 4 which is selected from the group consisting of bromide salts and sulphates. For preparing a pharmaceutical composition for the treatment of neoplastic pathology ,
Formula (1)
A compound represented by
In the formula (1), the R is
- saturated or unsaturated C ₂ -C _6, the linear or branched, and oxalic acid, malic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, diglycolic acid and citric acid A chain aliphatic polycarboxylic acid substituted on an aliphatic chain selected from the group consisting of :
- phthalic acid and 1,2,4-benzenetricarboxylic acid is selected from the group consisting, aryl polycarboxylic acids substituted at the aromatic ring;
-An amino acid selected from the group consisting of alanine, isoleucine, tyrosine, tryptophan and 4-aminobutanoic acid, which is an amino acid having an amino group at the α-position or other site ;
The acetal of the amino sugar daunosamine ;
- the compound from phosphoric acid is any free radicals formed inorganic acids and methods of use of these salts. Pharmaceutically acceptable, combined with therapeutic excipients and / or therapeutic diluents, and suitable for deriving and releasing the active ingredient at a controlled rate,
A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 5 as the active ingredient. 8. A pharmaceutical composition according to claim 7 suitable for administration by parenteral, intravenous, subcutaneous or intramuscular route. 8. The pharmaceutical composition according to claim 7 , which takes the form of granules, tablets, pills or capsules and is suitable for oral administration. 8. A pharmaceutical composition according to claim 7 suitable for topical and transdermal administration. The pharmaceutical composition according to claim 7 , which is suitable for purifying action during autologous bone marrow transplantation.